General description
As mentioned above, Rhodococcus Opacus can produce microbial lipids by accumulating aromatic compounds. Therefore, to focus on PET degradation and microbial lipid accumulation, the primary objective is to identify genes that link PET degradation and microbial lipid production. What kind of genes should we choose? After reviewing the literature, we used PETase and MHETase from Ideonella sakaiensis and a Tph operon from Comamonas testosterone to achieve our goal.
PETase: PETase was discovered in I. sakaiensis, which cleaves ester bonds in PET,hydrolyses polymerized PET molecules, and releases the dimer BHET and the monomer MHET.
The active site of PETase has an a-B-hydrolase fold, a twisted nine-stranded B-helix flanked by six a-helices, and the catalytic triad consists of Ser-His-Asp residues. Hydrolysis of the PET molecule involves a nucleophilic attack on the Ser residues of the PETase catalytic triad.
MHETase: MHETase has been found in I. sakaiensis, which cleaves the ester bond in MHET and degrades MHET to terephthalic acid TPA and ethylene glycol EG.
The active site of MHETase contains an a-B-hydrolase structure. The catalytic triad of Ser-His-Asp residues leads to the acylation-deacylation cycle in which a ping- pong mechanism catalyzes MHET. The Ser residues trigger a nucleophilic attack on the MHET molecule, forming an enzyme-acy intermediate that partially removes ethylene glycol from the molecule. After the glycol has been removed, deacylation begins when water molecules enter the active site. The end product, TPA, is then released.
The Tph operon from Comamonas testosterone YZW-D consists of a dioxygenase and a dehydrogenase enzyme. TphA2 and TphA3 are the oxygenase components, while TphA1 is the reductase component. All three together form a 1,2-dioxygenase that converts TPA to 1,2-dihydroxy-3, 5-cyclohexadiene-1,4-dicarboxylate (1,4-DCD).
1,4-DCD dehydrogenase includes TphB, which converts 1,4-DCD to PCA (protocatechuic acid).
Tph K encodes a regulatory factor involved in TPA catabolism.
According to the literature, Rhodococcus Opacus is more tolerant of ketones and aromatic compounds than engineered bacteria such as Escherichia coli and Bacillus subtilis. Therefore, Rhodococcus Opacus is a more suitable chassis organism for the degradation of PET plastics.
In addition, Rhodococcus Opacus PD630 is a natural oil producer that can accumulate a significant amount of microbial oil from a single carbon source, such as gluconate, glucose, or benzoate. The amount ranges from 50% to 75% of the cell's dry weight. In addition, Rhodococcus Opacus can use the substrate produced by the degradation of PET by PETase and MHE Tase to produce microbial oil, which can aid in the degradation and conversion of PET plastic waste. It is possible to degrade and transform PET plastic waste.
In conclusion, Rhodococcus opacus PD630 was selected as the chassis organism for degrading PET plastic and accumulating microbial oil production.